Etherlipid-containing multiple lipid liposomes

ABSTRACT

Described herein are liposomes containing etherlipids of the formula:as well as a phosphatidylcholine, a sterol, and a headgroup-derivatized lipid. These liposomes are useful in a variety of therapeutic regimens, including the treatment of cancers and inflammatory disorders.

This application is a continuation of our U.S. patent application Ser.No. 09/017,440, filed on Feb. 2, 1998, now U.S. Pat. No. 5,942,246,which is a continuation-in-part of U.S. patent application Ser. No.08/602,669, filed on Feb. 16, 1996, and which is now U.S. Pat. No.5,762,958.

Etherlipids are synthetic analogues of platelet activating factor (PAF;1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), an effector generallybelieved to be involved in a variety of physiological processes, such asinflammation, the immune response, allergic reactions and reproduction.Etherlipids have been shown to be effective antitumor agents in animals,and are believed to be selectively cytotoxic to a broad variety ofcancer cells (see, for example, Dietzfelbinger et al. (1993); Zeisig etal. (1993); Powis et al. (1990); Berdel (1991); Bhatia and Hadju (1991);Reed et al. (1991); Workman (1991); Workman et al. (1991); Bazill andDexter (1990); Berdel (1990); Counsell et al. (1990); Tritton andHickman (1990); Muschiol et al. (1990); Layton et al. (1980); Runge etal. (1980); Great Britain Patent No. 1,583,661; U.S. Pat. No.3,752,886). Etherlipids have also been shown to be antimetastatic andanti-invasive, and to be capable of cell differentiation induction.

Mechanisms of etherlipid cytotoxicity, while not definitivelyestablished, appear to involve action at, and possible disruption of,the cell membrane. The selective cytotoxicity of etherlipids may involveintracellular accumulation and differential activity of alkyl cleavageenzymes. Etherlipids may also be selective inhibitors ofphosphatidylinositol phospholipase C and protein kinase C activities, aswell as of phosphatidylcholine biosynthesis. Hence, etherlipids arepotentially quite useful as therapeutic agents. However, theiradministration can also lead to hemolysis, hepatic dysfunction andgastrointestinal disorders. Applicants have found that certain liposomalformulations of etherlipids can buffer these toxicities withoutinhibiting anticancer efficacy, and thereby can provide a moretherapeutically useful basis for etherlipid administration.

SUMMARY OF THE INVENTION

This invention provides a liposome comprising a bilayer having a lipidcomponent which comprises: (a) a phosphatidylcholine; (b) a sterol; (c)a headgroup derivatized lipid and, (d) an etherlipid. Theheadgroup-derivatized lipid, comprising a phosphatidylethanolaminelinked to a moiety selected from the group consisting of dicarboxylicacids, polyethylene glycols, gangliosides and polyalkylethers, comprisesfrom about 5 mole percent to about 20 mole percent of the bilayer'slipid component; the etherlipid comprises from about 10 mole percent toabout 30 mole percent of the lipid component.

Preferably, the phosphatidylcholine is dioleoyl phosphatidylcholine(“DOPC”), the sterol is cholesterol (“chol”), the headgroup-derivatizedlipid is dioleoyl phosphatidylethanolamine-glutaric acid (“DOPE-GA”) andthe etherlipid is

also known as “EL-18,” “ET-18-OCH₃,” or “edelfosine”). Most preferably,the liposome is a unilamellar liposome having a diameter of from greaterthan about 50 nm to less than about 200 nm, and the liposome's bilayerhas a lipid component comprising about 20 mole percent of theetherlipid, about 10 mole percent of the headgroup-derivatized lipid,about 30 mole percent cholesterol and about 40 mole percent dioleoylphosphatidylcholine.

Also provided herein is a pharmaceutical composition comprising apharmaceutically acceptable carrier and such liposomes. Further providedis a method of treating a mammal afflicted with a cancer, including, butnot limited to: a lung, brain, colon, ovarian or breast cancers, themethod comprising administering the pharmaceutical compositions of thisinvention to the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Time Course of Carboxyfluorescein Leakage from LiposomalEdelfosine Formulations Incubated at 48 deg. Celsius in PBS. ELL 28(uppermost curve, “ELL” indicating “etherlipid liposome”): Distearoylphosphatidylcholine (“DSPC”); cholesterol (“CHOL”); dioleoylphosphatidylethanolamine-glutaric acid (“DOPE-GA”); edelfosine “EL,”standing for “etherlipid” (the respective molar ratio of these lipidcomponents being 4:3:1:2); ELL 30 (second from top curve):EPC:CHOL:DOPE-GA:EL (4:3:1:2); ELL 25 (middle curve):DOPE:CHOL:DOPE-GA:EL (3:3:1:3); ELL 12 (second from bottom curve):DOPC:CHOL:DOPE-GA:EL (4:3:1:2); and, ELL 20 (bottom curve):DOPE:CHOL:DOPE-GA:EL (4:3:1:2). Y-axis: % CF Leakage; x-axis: time(seconds).

FIG. 2. Comparison of Hemolytic Activity and CF Leakage in EtherlipidLiposomes. From top-to-bottom: ELL 20 - ELL 12 - ELL 25 - ELL 30 - ELL28 (y=34231x^(−2.1614); R²=0.96). Y-axis: Hl₁₀; x-axis: % CF leakageupon incubation in PBS.

FIG. 3. Stability of Etherlipid Liposomal Formulations on Incubation in0.5% Serum at 37 Degrees Celsius. Y-axis: time (minutes); x-axis (fromleft-to-right): ELL 28, ELL 40, ELL 30; ELL 25; ELL 12; ELL 20. Inset:Y-axis: time (minutes); x-axis: ELL 28, ELL 40, ELL 30.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a liposome comprising a bilayer having a lipidcomponent which comprises: (a) a phosphatidylcholine; (b) a sterol; (c)a headgroup derivatized lipid containing a phosphatidylethanolamine anda moiety selected from the group consisting of dicarboxylic acids,gangliosides, polyethylene glycols and polyalkylethers, whichheadgroup-derivatized lipid comprises from about 5 mole percent to about20 mole percent of the bilayer's lipid component; and, (d) an etherlipidhaving the following formula:

the etherlipid comprising from greater than about 10 mole percent, toless than about 30 mole percent, of the bilayer's lipid component.

“Liposomes” are self-assembling structures comprising one or more lipidbilayers, each of which surrounds an aqueous compartment and comprisestwo opposing monolayers of amphipathic lipid molecules. Amphipathiclipids comprise a polar (hydrophilic) headgroup region covalently linkedto one or two non-polar (hydrophobic) acyl chains. Energeticallyunfavorable contacts between the hydrophobic acyl chains and the aqueousmedium are generally believed to induce lipid molecules to rearrangesuch that the polar headgroups are oriented towards the aqueous mediumwhile the acyl chains reorient towards the interior of the bilayer. Anenergetically stable structure is formed in which the acyl chains areeffectively shielded from coming into contact with the aqueous medium.

Liposomes can have a single lipid bilayer (unilamellar liposomes,“ULVs”), or multiple lipid bilayers (multilamellar liposomes, “MLVs”),and can be made by a variety of methods (for a review, see, for example,Deamer and Uster (1983)). These methods include without limitation:Bangham's methods for making multilamellar liposomes (MLVs); Lenk's,Fountain's and Cullis' methods for making MLVs with substantially equalinterlamellar solute distribution (see, for example, U.S. Pat. Nos.4,522,803, 4,588,578, 5,030,453, 5,169,637 and 4,975,282); andPapahadjopoulos et al.'s reverse-phase evaporation method (U.S. Pat. No.4,235,871) for preparing oligolamellar liposomes. ULVs can be producedfrom MLVs by such methods as sonication (see Papahadjopoulos et al.(1968)) or extrusion (U.S. Pat. No. 5,008,050 and U.S. Pat. No.5,059,421). The etherlipid liposome of this invention can be produced bythe methods of any of these disclosures, the contents of which areincorporated herein by reference.

Various methodologies, such as sonication, homogenization, French Pressapplication and milling can be used to prepare liposomes of a smallersize from larger liposomes. Extrusion (see U.S. Pat. No. 5,008,050) canbe used to size reduce liposomes, that is to produce liposomes having apredetermined mean size by forcing the liposomes, under pressure,through filter pores of a defined, selected size. Tangential flowfiltration (see WO89/008846), can also be used to regularize the size ofliposomes, that is, to produce liposomes having a population ofliposomes having less size heterogeneity, and a more homogeneous,defined size distribution. The contents of these documents areincorporated herein by reference. Liposome sizes can also be determinedby a number of techniques, such as quasi-electric light scattering, andwith equipment, e.g., Nicomp® particle sizers, well within thepossession of ordinarily skilled artisans.

The liposomes of this invention can be unilamellar or multilamellar.Preferably the liposomes are unilamellar and have diameters of less thanabout 200 nm, more preferably, from greater than about 50 nm to lessthan about 200 nm; such liposomes are preferably produced by a methodcomprising the steps of: dissolving lipids in a suitable organic solventso as to establish a lipidic solution; removing the organic solvent fromthe resulting lipidic solution; adding an aqueous solution so as to formliposomes; and, then extruding the resulting liposomes through asuitable filter.

Liposomes of the 50-200 nm size are preferred because they generallybelieved to circulate longer in mammals than do larger liposomes, whichare more quickly recognized by the mammals' reticuloendothelial systems(“RES”), and hence, more quickly cleared from the circulation. Longercirculation can enhance therapeutic efficacy by allowing more liposomesto reach their intended site of actions, e.g., tumors or inflammations.Small unilamellar liposomes, i.e., those generally less than 50 nm indiameter, carry amounts of bioactive agents which may be, in some cases,too low to be of sufficient therapeutic benefit.

R₁ of the etherlipid, the chain attached at the carbon #1 position ofits glycerol backbone by way of an oxygen, has the formula Y₁Y₂. Y₂ isCH₃ or CO₂H, but preferably is CH₃. Y₁ is—(CH₂)_(n1)(CH═CH)_(n2)(CH₂)_(n3)(CH═CH)_(n4)(CH₂)_(n5)(CH═CH)_(n6)(CH₂)_(n7)(CH═CH)_(n8)(CH₂)_(n9);the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 3 to 23;that is, the acyl chain is from 4-24 carbon atoms in length. n1 is equalto zero or is an integer of from 1 to 23; n3 is equal to zero or is aninteger of from 1 to 20; n5 is equal to zero or is an integer of from 1to 17; n7 is equal to zero or is an integer of from 1 to 14; n9 is equalto zero or is an integer of from 1 to 11; and each of n2, n4, n6 and 8is independently equal to zero or 1.

The hydrocarbon chain is preferably saturated, that is, it preferablyhas no double bonds between adjacent carbon atoms, each of n2, n4, n6and n8 then being equal to zero. Accordingly, Y₁ is preferably(CH₂)_(n1). More preferably, R₁ is (CH₂)_(n1)CH₃, and most preferably,is (CH₂)₁₇CH₃. Alternatively, the chain can have one or more doublebonds, that is, it can be unsaturated, and one or more of n2, n4, n6 andn8 can be equal to 1. For example, when the unsaturated hydrocarbon hasone double bond, n2 is equal to 1, n4, n6 and n8 are each equal to zeroand Y₁ is (CH₂)_(n1)CH═CH(CH₂)_(n3). n1 is then equal to zero or is aninteger of from 1 to 21, and n3 is also zero or is an integer of from 1to 20, at least one of n1 or n3 not being equal to zero.

Z is oxygen, sulfur, NH, or —NHC(O)—, Z then being connected to themethyl group by way of either the nitrogen or carbonyl carbon. Z canalso be —OC(O)—, it then being connected to the methyl group by way ofeither the oxygen or carbonyl carbon atom. Preferably, Z is O;accordingly, this invention's glycerol-based etherlipids preferably havea methoxy group at the sn-2 position of their glycerol backbone.

R₂ is an alkyl group, or a halogen-substituted alkyl group, having theformula (C(X₁)_(n10)(X₂)_(n11))_(n12)CX₃X₄X₅, wherein each of X₁, X₂,X₃, X₄, and X₅ is independently hydrogen or a halogen, but is preferablyhydrogen. n10 is equal to zero, 1 or 2; n11 is equal to zero, 1, or 2;and n12 is equal to zero or an integer of from 1 to 23, but is mostpreferably, zero, R₂ then being CX₃X₄X₅. X₃, X₄, and X₅ are mostpreferably H, R₂ then being CH₃. Accordingly, the etherlipid preferablyhas a methyl group attached to its carbon #2. However, R₂ can then alsobe CH₂F, CHF₂ or CF₃. When n12 is not zero, the sum of n10+n11 is equalto 2, n12 is preferably equal to 1, and R₂ is preferably CH₂CH₃, CH₂CF₃or CF₂CF₃.

Most preferably, the etherlipid is one in which Y₂ is CH₃, R₁ is(CH₂)_(n1)CH₃, R₂ is CH₃ and Z is O. The preferred etherlipid istherefore:

that is, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine(“ET-18-OCH₃” or “edelfosine”).

Preferably, the phosphatidylcholine (“PC”) is partially or whollyunsaturated, that is, it has two acyl chains, at least one of which hasat least one double bond between adjacent carbon atoms. More preferably,presently, the PC is dioleoyl phosphatidylcholine (“DOPC”). Theliposome's lipid bilayer also contains a sterol, which generally affectsthe fluidity of lipid bilayers (see, for example, Lewis and McElhaney(1992) and Darnell et al. (1986)) Accordingly, sterol interactions withsurrounding hydrocarbon chains generally inhibit emigration of thesechains from the bilayer. The sterol of the liposomes of this inventionis preferably, but not necessarily, cholesterol, and can also be avariety of other sterolic compounds.

A “headgroup-derivatized” lipid is a lipid which, when present in aliposomal lipid bilayer with an etherlipid, can buffer the toxicity ofthe etherlipid. That is, the derivatized lipid can decrease theetherlipid's toxicity, such that it is generally less toxic than thefree form of the etherlipid. Headgroup-derivatized lipids generally areamphipathic lipids comprising hydrophobic acyl chains, and aphosphorylethanolamine group to which a suitable chemical moiety hasbeen attached. Acyl chains are those which can adopt compatible packingconfigurations with the hydrophobic portions of other lipids present inthe bilayer, and which can interact with an etherlipid such that releaseof the etherlipid from the bilayer is inhibited and etherlipid toxicityis buffered; these are saturated or unsaturated, straight-chained orbranched, and typically contain from 4 to 24 carbon atoms in a straightchain. Preferred acyl chains are palmitate or oleate chains; hencepreferred headgroup-modified lipids are dipalmitoylphosphatidylethanolamine (“DPPE”), palmitoyloleoylphosphatidylethanolamine (“POPE”) or dioleoyl phosphatidylethanolamine(“DOPE”); most preferably, presently, the lipid is DOPE.

Chemical moieties suitable for attachment to such lipids are those, suchas dicarboxylic acids, gangliosides, polyethylene glycols, polyalkylethers and the like, which can be attached to the amino group of aphosphorylethanolamine, and which give rise to lipids having toxicitybuffering, circulation-enhancing properties. Means of identifyingsuitable chemical moieties, for example by subjecting derivatized lipidsto in vitro and in vivo toxicity testing, are well known to, and readilypracticed by, ordinarily skilled artisans given the teachings of thisinvention. Means of attaching chemical moieties tophosphorylethanolamine groups are also well known to, and readilypracticed by, ordinarily skilled artisans.

Toxicity buffering capacities of headgroup-derivatized lipids can bedetermined by a number of in vitro and in vivo testing methods wellknown to, and readily practiced by, ordinarily skilled artisans, giventhe teachings of this invention. For example, etherlipid-induced redblood cell (RBC) hemolysis can be examined in vitro by combining anetherlipid with an RBC suspension, incubating the combination, and thenquantitating the percentage of RBC lysis.

Toxicity-buffering can also be assessed by determining the etherlipid'stherapeutic window “TW,” which is a numerical value derived from therelationship between the compound's induction of hemolysis and itsability to inhibit the growth of tumor cells. TW values are determinedin accordance with the formula Hl₅/Gl₅₀ (wherein “Hl₅” equals theconcentration of compound inducing the hemolysis of 5% of the red bloodcells in a culture, and wherein “Gl₅₀” equals the dose of compoundinducing fifty percent growth inhibition in a population of cellsexposed to the agent). The higher an agent's Hl₅ value, the lesshemolytic is the agent—higher Hl₅'s mean that greater concentrations ofcompound are required to be present in order for the compound to induce5% hemolysis. Hence, the higher its Hl₅, the more therapeuticallybeneficial is a compound, because more of it can be given beforeinducing the same amount of hemolysis as an agent with a lower Hl₅. Bycontrast, lower Gl₅₀'s indicate better therapeutic agents—a lower Gl₅₀value indicates that a lesser concentration of an agent is required for50% growth inhibition. Accordingly, the higher is its Hl₅ value and thelower is its Gl₅₀ value, the better are a compound's agent's therapeuticproperties.

Generally, when a bioactive agent's TW is less than 1, it cannot be usedeffectively as a therapeutic agent. That is, the agent's Hl₅ value issufficiently low, and its Gl₅) value sufficiently high, that it isgenerally not possible to administer enough of the agent to achieve asufficient level of tumor growth inhibition without also attaining anunacceptable level of hemolysis. Etherlipid liposomes having bilayersthat also comprise headgroup-derivatized lipids have TW's of greaterthan 1. Preferably, the TW of an etherlipid in a liposomal bilayer alsocomprising a headgroup-derivatized lipid is greater than about 1.5, morepreferably, greater than about 2, and still more preferably, greaterthan about 3.

Headgroup-derivatized lipids can also be circulation-enhancing lipids,that is, the modifications directed to lipid toxicity buffering can alsoafford circulation enhancement. Accordingly, headgroup-derivatizedlipids can inhibit clearance of liposomes from the circulatory systemsof animals to which they have been administered. Liposomes are generallybelieved to be cleared from an animal's body by way of itsreticuloendothelial system (RES). Avoiding RES clearance means that thefrequency of liposome administration can be reduced, and that less of aliposome-associated bioactive agent need be administered to achievedesired serum levels of the agent. Enhanced circulation times can alsoallow targeting of liposomes to non-RES containing tissues.

Liposome outer surfaces are believed to become coated with serumproteins, such as opsonins, in animals' circulatory systems. Withoutintending in any way to be limited by theory, it is believed thatliposome clearance can be inhibited by modifying the outer surface ofliposomes such that binding of serum proteins thereto is generallyinhibited. Effective surface modification, that is, alterations to theouter surfaces of liposomes which result in inhibition of opsonizationand RES uptake, is believed to be accomplished by incorporating intoliposomal bilayers lipids whose polar headgroups have been derivatizedby attachment thereto of a chemical moiety which can inhibit the bindingof serum proteins to liposomes such that the pharmacokinetic behavior ofthe liposomes in the circulatory systems of animals is altered (see,e.g., Blume et al. (1993); Gabizon et al. (1993); Park et al. (1992);Woodle et al. U.S. Pat. No. 5,013,556; and, U.S. Pat. No. 4,837,028).

Presently, dicarboxylic acids, such as glutaric, sebacic, succinic andtartaric acids, are preferred components of headgroup-derivatizedlipids. Most preferably, the dicarboxylic acid is glutaric acid (“GA”).Accordingly, preferred headgroup-derivatized lipids includephosphatidylethanolamine-dicarboxylic acids such as dipalmitoylphosphatidylethanolamine-glutaric acid (“DPPE-GA”), palmitoyloleoylphosphatidylethanolamine-glutaric acid (“POPE-GA”) and dioleoylphosphatidylethanolamine-glutaric acid (“DOPE-GA”). Most preferably,presently, the derivatized lipid is DOPE-GA.

The liposomes of this invention can comprise one or more additionallipids, that is, lipids in addition to the phosphatidylcholine, sterol,headgroup-derivatized lipid and etherlipid already present in theliposomes' bilayers. Additional lipids are selected for their ability toadapt compatible packing conformations with the other lipid componentsof the bilayer such that the lipid constituents are tightly packed, andrelease of the lipids from the bilayer is inhibited. Lipid-based factorscontributing to compatible packing conformations are well known toordinarily skilled artisans and include, without limitation, acyl chainlength and degree of unsaturation, as well as the headgroup size andcharge. Accordingly, suitable additional lipids, including variousphosphatidylethanolamines (“PE's”) such as egg phosphatidylethanolamine(“EPE”) or dioleoyl phosphatidylethanolamine (“DOPE”) can be selected byordinarily skilled artisans without undue experimentation.

Preferred embodiments of this invention have the phosphatidylcholinebeing DOPC, the sterol being cholesterol (“chol”), theheadgroup-derivatized lipid being DOPE-GA and the etherlipid beingET-18-OCH₃. Most preferably, presently, the liposome comprises DOPC,chol, DOPE-GA and ET-18-O-CH₃ in a respective molar ratio of 4:3:1:2,wherein DOPC comprises 40 mole % of the bilayer lipid component, chol30% mole, DOPE-GA 10 mole % and the etherlipid 20 mole %. Preferably,the liposomes are unilamellar and have an average diameter of from about50 nm to about 200 nm, “average” meaning that the median diameter of apopulation of this invention's liposomes is between about 50 and 200 nm.

The liposome can comprise an additional bioactive agent, that is, abioactive agent in addition to the etherlipid. A “bioactive agent” isany compound or composition of matter that can be administered toanimals, preferably humans. Such agents can have biological activity inanimals; the agents can also be used diagnostically in the animals.Bioactive agents which may be associated with liposomes include, but arenot limited to: antiviral agents such as acyclovir, zidovudine and theinterferons; antibacterial agents such as aminoglycosides,cephalosporins and tetracyclines; antifungal agents such as polyeneantibiotics, imidazoles and triazoles; antimetabolic agents such asfolic acid, and purine and pyrimidine analogs; antineoplastic agentssuch as the anthracycline antibiotics and plant alkaloids; sterols suchas cholesterol; carbohydrates, e.g., sugars and starches; amino acids,peptides, proteins such as cell receptor proteins, immunoglobulins,enzymes, hormones, neurotransmitters and glycoproteins; dyes;radiolabels such as radioisotopes and radioisotope-labeled compounds;radiopaque compounds; fluorescent compounds; mydriatic compounds;bronchodilators; local anesthetics; and the like.

Liposomal bioactive agent formulations can enhance the therapeutic indexof the bioactive agent, for example by buffering the agent's toxicity.Liposomes can also reduce the rate at which a bioactive agent is clearedfrom the circulation of animals. Accordingly, liposomal formulation ofbioactive agents can mean that less of the agent need be administered toachieve the desired effect. Additional bioactive agents preferred forthe liposome of this invention include antimicrobial, anti-inflammatoryand antineoplastic agents, or therapeutic lipids, for example,ceramides. Most preferably, the additional bioactive agent is anantineoplastic agent.

Liposomes can be loaded with one or more biologically active agents bysolubilizing the agent in the lipid or aqueous phase used to prepare theliposomes. Alternatively, ionizable bioactive agents can be loaded intoliposomes by first forming the liposomes, establishing anelectrochemical potential, e.g., by way of a pH gradient, across theoutermost liposomal bilayer, and then adding the ionizable agent to theaqueous medium external to the liposome (see Bally et al. U.S. Pat. No.5,077,056 and WO86/01102).

The liposome of this invention can be dehydrated, stored and thenreconstituted such that a substantial portion of its internal contentsare retained. Liposomal dehydration generally requires use of ahydrophilic drying protectant such as a disaccharide sugar at both theinside and outside surfaces of the liposome bilayers (see U.S. Pat. No.4,880,635). This hydrophilic compound is generally believed to preventthe rearrangement of the lipids in the liposome, so that the size andcontents are maintained during the drying procedure and throughsubsequent rehydration. Appropriate qualities for such dryingprotectants are that they be strong hydrogen bond acceptors, and possessstereochemical features that preserve the intramolecular spacing of theliposome bilayer components. Alternatively, the drying protectant can beomitted if the liposome preparation is not frozen prior to dehydration,and sufficient water remains in the preparation subsequent todehydration.

Also provided herein is a pharmaceutical composition comprising apharmaceutically acceptable carrier and the liposome of this invention.“Pharmaceutically acceptable carriers” as used herein are those mediagenerally acceptable for use in connection with the administration oflipids and liposomes, including liposomal bioactive agent formulations,to animals, including humans. Pharmaceutically acceptable carriers aregenerally formulated according to a number of factors well within thepurview of the ordinarily skilled artisan to determine and account for,including without limitation: the particular liposomal bioactive agentused, its concentration, stability and intended bioavailability; thedisease, disorder or condition being treated with the liposomalcomposition; the subject, its age, size and general condition; and thecomposition's intended route of administration, e.g., nasal, oral,ophthalmic, topical, transdermal, vaginal, subcutaneous, intramammary,intraperitoneal, intravenous, or intramuscular (see, for example, Nairn(1985)). Typical pharmaceutically acceptable carriers used in parenteralbioactive agent administration include, for example, D5W, an aqueoussolution containing 5% weight by volume of dextrose, and physiologicalsaline. Pharmaceutically acceptable carriers can contain additionalingredients, for example those which enhance the stability of the activeingredients included, such as preservatives and anti-oxidants.

Further provided is a method of treating a mammal afflicted with acancer, e.g., a brain, breast, lung, colon or ovarian cancer, or aleukemia, lymphoma, sarcoma, carcinoma, which comprises administeringthe pharmaceutical composition of this invention to the mammal,etherlipids being believed to be selectively cytotoxic to tumor cells.Generally, liposomal etherlipids can be used to treat cancers treatedwith free, that is, nonliposomal, etherlipids. However, encapsulation ofan etherlipid in a liposome can enhance its therapeutic index, andtherefore make the liposomal etherlipid a more effective treatment.

An amount of the composition comprising an anticancer effective amountof the etherlipid, typically from about 0.1 to about 1000 mg of thelipid per kg of the mammal's body, is administered, preferablyintravenously. For the purposes of this invention, “anticancer effectiveamounts” of liposomal etherlipids are amounts effective to inhibit,ameliorate, lessen or prevent establishment, growth, metastasis orinvasion of one or more cancers in animals to which the etherlipids havebeen administered. Anticancer effective amounts are generally chosen inaccordance with a number of factors, e.g., the age, size and generalcondition of the subject, the cancer being treated and the intendedroute of administration, and determined by a variety of means, forexample, dose ranging trials, well known to, and readily practiced by,ordinarily skilled artisans given the teachings of this invention.Antineoplastic effective amounts of the liposomal etherlipid of thisinvention are about the same as such amounts of free, nonliposomal,etherlipids, e.g., from about 0.1 mg of the etherlipid per kg of bodyweight of the mammal being treated to about 1000 mg per kg.

Preferably, the liposome administered is a unilamellar liposome havingan average diameter of from about 50 nm to about 200 nm. The anti-cancertreatment method can include administration of one or more bioactiveagents in addition to the liposomal etherlipid, these additional agentspreferably, but not necessarily, being included in the same liposome asthe etherlipid. The additional bioactive agents, which can be entrappedin liposomes' internal compartments or sequestered in their lipidbilayers, are preferably, but not necessarily, anticancer agents orcellular growth promoting factors.

The liposomes are also effective as anti-inflammatory agents.

This invention will be better understood from the following examples.However, those of ordinary skill in the art will readily understand thatthese examples are merely illustrative of the invention as defined inthe claims which follow thereafter.

EXAMPLES Example 1 Preparation

Liposomes were prepared with edelfosine (ET-18-O-CH₃, 5 mg/ml), variousother lipids obtained from Avanti Polar Lipids, Birmingham, Ala., andcholesterol (Sigma Chemical Co.). Briefly, the lipids were dissolved inan organic solvent, such as chloroform, at various mole ratios. Theorganic solvent was then removed, and the dried lipids were rehydrated,e.g., with Dulbecco's phosphate-buffered saline (D-PBS) (Gibco BRL LifeTechnologies, Grand Island, N.Y.). The resulting liposomes were extrudedthrough 0.1 micron Nuclepore® filters (see, for example, Mayer et al.,1985). Liposome sizes were then determined by light scattering, using aNicomp® Model 370 Submicron Particle Sizer.

Example 2 Red Blood Cell (“RBC”) Hemolysis Assay

A 4% suspension of red blood cells (RBCs), 0.5 ml, was washed threetimes in PBS and then incubated with free (non-liposomal) etherlipid orliposomal etherlipid, prepared as described above. These samples werevortexed on a 37 deg. C. agitator for 20 hours, and were thencentrifuged for 10 minutes at 3000 rpm. 0.2 ml of the resultingsupernatant was diluted to 1 ml with water, and the percentage hemolysisin the sample was quantitated by spectrophotometric examination at 550nm.

Results from these studies are presented in Table 1 (see below), whereinthe concentration (μM) of edelfosine required to cause 10 % RBChemolysis (“Hl₁₀”) in each formulation is set forth. The table's firstcolumn is a short-hand designation of the particular formulation, “ELL”standing for “etherlipid liposome.” The second column indicates thecomponents of the formulation tested, including dioleoylphosphatidylethanolamine “(DOPE”), cholesterol (“CHOL”),dioleoyl-phosphatidylethanolamine-glutaric acid (“DOPE-GA”), dioeloylphosphatidylcholine (“DOPC”), palmitoyloleoyl phosphatidylcholine(“POPC”), distearoyl phosphatidylcholine (“DSPC”), eggphosphatidylcholine (“EPC”) and edelfosine (“EL,” for etherlipid). Therespective molar ratios of the various lipid components are also setforth. The last row of the table gives the Hl₁₀ value for edelfosinealone, i.e., not incorporated in a liposome.

TABLE 1 Formulation Composition HI₁₀ ELL 20 DOPE:CHOL:DOPE-GA:EL 1726 ±160  4    3     1     2 ELL 12 DOPC:CHOL:DOPE-GA:El 670 ± 60 4    3     1     2 ELL 40 POPC:CHOL:DOPE-GA:EL 65 ± 6 4    3     1     2 ELL 28 DSPC:CHOL:DOPE-GA:EL 32 ± 3 4    3     1     2 ELL 25 DOPE:CHOL:DOPE-GA:EL 537 ± 50 4    3     1     2 ELL 30 EPC:CHOL:DOPE-GA:EL 314 ± 30 4    3     1     2 Edelfosine        — 5 ± 1

Example 3 Fluorescence Spectroscopy

Liposomes were prepared as described above, and in the presence of anaqueous solution of 0.1 M 6-carboxyfluorescein (“CF”); free CF was thenremoved by gel filtration. CF efflux from liposomes over time wasmonitored by measuring, at 520 nm (excitation at 490 nm), increases inCF fluorescence in the aqueous phase external to the liposomes, upontheir incubation in PBS at 48 deg. C. Fluorescence values, presented inFIG. 1 herein, are expressed as a percentage increase in CF fluorescencerelative to the total CF fluorescence found after disrupting liposomeswith Triton X-100.

FIG. 2 herein compares hemolytic activity and CF leakage in variousliposomal formulations described in Table 1, upon incubation of theliposomes in PBS at 48 deg. C. for 25 minutes. FIG. 3 compares the timerequired for 50% CF leakage in various liposomal formulations, upontheir incubation in 0.5% serum at 37 deg. C.

References Cited U.S. Patent Documents

U.S. Pat. Nos. 4,159,988, 4,163,748, 4,235,871, 4,382,035, 4,522,803,4,588,578, 4,734,225, 4,804,789, 4,837,028, 4,920,016, 4,975,282,5,008,050, 5,013,556, 5,030,453, 5,059,421, 5,077,056, 5,169,637,3,752,886

Foreign Patent Documents

WO89/008846, 1,583,661, 4,132,345

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What is claimed is:
 1. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a liposome, the liposomecomprising: a lipid bilayer which comprises: (a) a phosphatidylcholine;(b) a sterol; (c) a headgroup derivatized lipid comprising aphosphatidylethanolamine linked at the ethanolamine group to a moietyselected from the group consisting of dicarboxylic acids, polyethyleneglycols, gangliosides and polyalkyl ethers; and, (d) an etherlipidhaving the formula:

wherein: R₁ is Y₁Y₂, Y₂ is CH₃ or CO₂H, Y₁ is(CH₂)_(n1)(CH═CH)_(n2)(CH₂)_(n3)(CH═CH)_(n4)(CH₂)_(n5)(CH═CH)_(n6)(CH₂)_(n7)(CH═CH)_(n8)(CH₂)₉, the sum of n₁+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is aninteger of from 3 to 23, n1 is equal to zero or is an integer of from 1to 23, n3 is equal to zero or is an integer of from 1 to 20, n5 is equalto zero or is an integer of from 1 to 17, n7 is equal to zero or is aninteger of from 1 to 14, n9 is equal to zero or is n integer of from 1to 11, and each of n2, n4, n6 and n8 is independently zero or 1; Z isoxygen or sulfur; and, the headgroup-derivatized lipid comprises fromabout 5 mole percent to about 20 mole percent of the liposome's lipidbilayer and the etherlipid comprises from greater than about 10 molepercent to less than about 30 mole percent of the bilayer.
 2. A methodof treating a mammal afflicted with a cancer selected from the groupconsisting of lung cancers, brain cancers, colon cancers, ovariancancers, breast cancers, leukemias, lymphomas, sarcomas and carcinomas,the method comprising administering to the mammal an amount of thepharmaceutical composition of claim 1 which comprises from about 0.1 mgof the etherlipid per kg of body weight of the mammal to about 1000 mgper kg.
 3. The method of claim 2, comprising administering to the mammalan additional biologically active agent.
 4. The method of claim 3,wherein the additional agent is selected from the group consisting ofantineoplastic agents, antimicrobial agents, therapeutic lipids andhematopoietic cell growth stimulating agents.
 5. The method of claim 2,wherein the liposome is a unilamellar liposome having a diameter of fromabout 50 nm to about 200 nm.